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Pierre Emmanuel Milhiet

Publications and source records attributed to Pierre Emmanuel Milhiet.

8 recordsLinked to original sources
PubMed2004

Interaction of primary amphipathic cell-penetrating peptides with phospholipid-supported monolayers.

The mesoscopic organization adopted by two primary amphipathic peptides, P(beta) and P(alpha), in Langmuir-Blodgett (LB) films made of either the pure peptide or peptide-phospholipid mixtures was examined by atomic force microscopy. P(beta), a potent cell-penetrating peptide (CPP), and P(alpha) mainly differ by their conformational states, predominantly a beta-sheet for P(beta) and an alpha-helix for P(alpha), as determined by Fourier transform infrared spectroscopy. LB films of pure peptide, transferred significantly below their collapse pressure, were characterized by the presence of supramolecular structures, globular aggregates for P(beta) and filaments for P(alpha), inserted into the monomolecular film. In mixed peptide-phospholipid films, similar structures could be observed, as a function of the phospholipid headgroup and acyl chain saturation. They often coexisted with a liquid-expanded phase composed of miscible peptide-lipid. These data strongly suggest that primary amphipathic CPP and antimicrobial peptides may share, to some extent, common mechanisms of interaction with membranes.

Membranes, Artificial↗
PubMed2004

Use of cyclodextrin for AFM monitoring of model raft formation.

The lipid rafts membrane microdomains, enriched in sphingolipids and cholesterol, are implicated in numerous functions of biological membranes. Using atomic force microscopy, we have examined the effects of cholesterol-loaded methyl-beta-cyclodextrin (MbetaCD-Chl) addition to liquid disordered (l(d))-gel phase separated dioleoylphosphatidylcholine (DOPC)/sphingomyelin (SM) and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC)/SM supported bilayers. We observed that incubation with MbetaCD-Chl led to the disappearance of domains with the formation of a homogeneously flat bilayer, most likely in the liquid-ordered (l(o)) state. However, intermediate stages differed with the passage through the coexistence of l(o)-l(d) phases for DOPC/SM samples and of l(o)-gel phases for POPC/SM bilayers. Thus, gel phase SM domains surrounded by a l(o) matrix rich in cholesterol and POPC could be observed just before reaching the uniform l(o) state. This suggests that raft formation in biological membranes could occur not only via liquid-liquid but also via gel-liquid immiscibility. The data also demonstrate that MbetaCD-Chl as well as the unloaded cyclodextrin MbetaCD make holes and preferentially extract SM in supported bilayers. This strongly suggests that interpretation of MbetaCD and MbetaCD-Chl effects on cell membranes only in terms of cholesterol movements have to be treated with caution.

Cholesterol↗
PubMed2003

AFM imaging of lipid domains in model membranes.

Characterization of the two-dimensional organization of biological membranes is one of the most important issues that remains to be achieved in order to understand their structure-function relationships. According to the current view, biological membranes would be organized in in-plane functional microdomains. At least for one category of them, called rafts, the lateral segregation would be driven by lipid-lipid interactions. Basic questions like the size, the kinetics of formation, or the transbilayer organization of lipid microdomains are still a matter of debate, even in model membranes. Because of its capacity to image structures with a resolution that extends from the molecular to the microscopic level, atomic force microscopy (AFM) is a useful tool for probing the mesoscopic lateral organization of lipid mixtures. This paper reviews AFM studies on lateral lipid domains induced by lipid-lipid interactions in model membranes.

Animals↗
PubMed2003

[Atomic force microscopy: from cellular imaging to molecular manipulation].

Using a sharp tip attached at the end of a soft cantilever as a probe, the atomic force microscope (AFM) explores the surface topography of biological samples bathed in physiological solutions. In the last few years, the AFM has gained popularity among biologists. This has been obtained through the improvement of the equipment and imaging techniques as well as through the development of new non-imaging applications. Biological imaging has to face a main difficulty that is the softness and the dynamics of most biological materials. Progress in understanding the AFM tip-biological samples interactions provided spectacular results in different biological fields. Recent examples of the possibilities offered by the AFM in the imaging of intact cells, isolated membranes, membrane model systems and single molecules at work are discussed in this review. Applications where the AFM tip is used as a nanotool to manipulate biomolecules and to determine intra- and intermolecular forces from single molecules are also presented.

Animals↗
PubMed2002

Dominant negative effectors of heparin affin regulatory peptide (HARP) angiogenic and transforming activities.

Heparin affin regulatory peptide (HARP) is an heparin-binding growth factor, highly expressed in several primary human tumors and considered as a rate-limiting angiogenic factor in tumor growth, invasion, and metastasis. Implication of this protein in carcinogenesis is linked to its mitogenic, angiogenic, and transforming activities. Recently, we have demonstrated that the C-terminal residues 111-136 of HARP are required for its mitogenic and transforming activities (Bernard-Pierrot, I., Delbe, J., Caruelle, D., Barritault, D., Courty, J., and Milhiet, P. E. (2001) J. Biol. Chem. 276, 12228-12234). In this paper, HARP deleted of its last 26 amino acids was shown to act as a dominant negative effector for its mitogenic, angiogenic, transforming, and tumor-formation activities by heterodimerizing with the wild type protein. Similarly, the synthetic corresponding peptide P111-136 displayed in vitro inhibition of wild type HARP activities, but in this case, the inhibition was mainly explained by the competition of the peptide with HARP for the binding to the extracellular domain of the high affinity ALK receptor.

3T3 Cells↗
PubMed2002

Immunoassay for measuring the heparin-binding growth factors HARP and MK in biological fluids.

Heparin-affin regulatory peptide (HARP) and Midkine (MK) belong to a family of growth/differentiation factors that have a high affinity for heparin. The involvement of these molecules in various proliferative diseases prompted us to develop an assay for measuring the concentrations of these factors in biological fluids and culture media. This report describes an immunoassay that uses only commercially available materials, based on the high affinity of certain molecules for heparin. It consists of adsorbing heparin-BSA covalent complexes to microtiter plate wells and to quantify the heparin bound HARP or MK by using appropriate antibody. The method is specific and measures concentrations ranging from 40-1200 pg/mL HARP and from 25-1200 pg/mL MK and various parameters are investigated. The within-assay coefficient of variation was less than 5% for both assays. The method was checked by measuring the concentrations of these growth factors in the sera of healthy humans and in patients with cancer. As previously reported, we confirmed that the serum concentrations of MK are higher in patients with tumours (n = 139) than in controls (n = 19). The synthesis of HARP and MK by various cells in culture was also analysed.

Adult↗
PubMed2001

Cholesterol is not crucial for the existence of microdomains in kidney brush-border membrane models.

The external membrane leaflet plays a key role in the organization of the cell plasma membrane as a mosaic of ordered microdomains enriched in sphingolipids and cholesterol and of fluid domains. In this study, the thermotropic behavior and the topology of bilayers made of a phosphatidylcholine/sphingomyelin mixture, which mimicks the lipid composition of the external leaflet of renal brush-border membranes, were examined by differential scanning calorimetry and atomic force microscopy. In the absence of cholesterol, a broad phase separation process occurred where ordered gel phase domains of size varying from the mesoscopic to the microscopic scale, enriched in sphingomyelin, occupied half of the bilayer surface at room temperature. Increasing amounts of cholesterol progressively decreased the enthalpy of the transition and modified the topology of membranes domains up to a concentration of 33 mol % for which no membrane domains were detected. These results strongly suggest that, in membranes highly enriched in sphingolipids like renal and intestinal brush borders, there is a threshold close to the physiological concentration above which cholesterol acts as a suppressor rather than as a promoter of membrane domains. They also suggest that cholesterol depletion does not abolish the lateral heterogenity in brush-border membranes.

Animals↗